The invention relates to novel hirudin muteins and to hirudin/polyalkylene glycol conjugates thereof, to the preparation thereof and to the use thereof both for the prophylaxis and therapy of cardiovascular disorders and for modification of macromolecular carriers.
Hirudin is a naturally occurring protein with anticoagulant properties which has been known for a long time. It is the most potent and most selective thrombin inhibitor yet known (Naturwissenschaften, (1955) 42,537; Hopppe-Seylers Z. fur Biol. Chemie, (1985) 366, 379). The polypeptide can be isolated from the medical leech Hirudo medicinalis and is composed of 65 amino acids, contains three disulfide bridges and is sulfated at position tyrosine 63. In addition, there also exist several naturally occurring isoforms which differ from the original hirudin by amino-acid replacement in various positions (Folia Haematol. (1988), 115, 30). Likewise, variants prepared by genetic engineering are known (Biochemistry (1988), 27, 6517, FEBS-Lett. (1988), 229, 87). Hirudin and various variants can now be obtained by genetic engineering means, the sulfate residue on amino acid Tyr 63 being absent in hirudins prepared by genetic engineering methods (Biochemistry (1989), 28, 2941, DNA (1986), 5, 511). The good physiological tolerability of this coagulation inhibitor has likewise been known for some time (Pharmazie (1981), 10, 653).
Despite its favorable pharmacodynamic properties, hirudin is, by reason of its low half-life in the blood of about 50 min., little suited to long-lasting therapeutic applications. It is known that the half-life of proteins can be extended by conjugation with macro-molecules (J. Biol. Chem. (1977), 252, 3582; Biochim. Biophys. Acta (1981), 660, 293). It is often observed after a derivatization of this type with, for example, polyethylene glycol that there is a significant deterioration in the enzymatic activity, which greatly restricts the utilizability of such modified proteins (Cancer. Treat. Rep. (1979), 63, 1127; Chemistry Lett. (1980), 773). In the case of hirudin, it has recently been shown by Walsmann that it was possible by coupling to dextran to achieve a distinct extension of the half-life from about 50 min. to more than 7 h, although there was a drastic loss of activity (Pharmazie (1989), 44, 72). Therapeutic use of such dextan-hirudins [sic] is, despite the favorable alteration in the half-life, impeded by the very low yield of product, the drastically reduced specific activity and the changes, which are possibly connected therewith, in the pharmacodynamic properties.
Conjugation of proteins to macromolecules is often achieved by reaction of the carboxyl groups of the amino acids aspartic acid or glutamic acid, by reaction of the sulfhydryl group of the amino acid cystein or by reaction of the side-chain amino group of the amino acid lysine in the relevant protein. However, it is often precisely the said amino acids which are essentially important for the function of the relevant protein. The derivatization of a protein may be associated with a change in the physical/chemical or enzymatic properties, even up to inactivation. Hirudin contains several aspartic acid and glutamic acid residues, mainly in the C-terminal region of the molecule. Lysine residues, are located in position 27, 36 and 47 in the hirudin molecule. Furthermore, coupling via the C terminus or the N terminus of the molecule would be conceivable. However, it is known that both the acidic amino acids in the C-terminal region (FEBS. Lett. (1983), 164 307-313) and the basic lysine residues, especially the lysine residue No. 47 which is highly exposed in the molecule, are crucially involved in the interaction of hirudin with the protease thrombin (Biol. Chem. Hoppe-Seyler (1985), 366, 379-385). Reactions at the N terminus, such as, for example, an extension (Biochemistry (1989), 28, 10079) lead to a drastic decrease in the inhibitory activity of hirudin. It was therefore not to be expected that derivatization of hirudin can be achieved without significant loss of activity. This expectation is distinctly verified by the work carried out by Walsmann (Pharmazie (1989), 44, 72) on the derivatization of the lysine residues of hirudin with dextran.
Because of the large number of acidic amino acids, conjugation of macromolecules with the carboxyl side-chains of hirudin is not expected to give a pure product. Even if only the basic functionalities of the polypeptide are derivatized, a mixture of up to 32 different compounds is expected. In the case of the mono-, di- and trisubstituted derivatives, a large number of positional isomers is conceivable, and these have substantially the same physical and chemical properties but differ an their biological activity. Even if the majority of the theoretically conceivable conjugates make contributions only in the trace range to the overall mixture, there must be expected to be considerable problems in the separation of an inhomogeneous product.